Presynaptic monoamine reuptake is a major factor that influences extraneuronal monoamine levels and terminates synaptic transmission. Monoamine transporters within the SL6 carrier gene family mediate reuptake and are the molecular targets for the addictive psychostimulants cocaine, amphetamines, and MDMA (ecstasy), as well as for the therapeutic agents: methylphenidate (Ritalin) and buproprion (Wellbutrin, Zyban). These agents potently inhibit monoamine transport, significantly elevating extracellular monoamine levels and enhancing downstream signaling. Thus, transporter cell surface availability is paramount to both normal synaptic transmission and psychoactive drug efficacy. Abundant evidence demonstrates that monoamine transporters dynamically traffic to and from the plasma membrane. Moreover, both amphetamine exposure and protein kinase C (PKC) activation change transporter surface availability by modulating transporter trafficking kinetics. Although regulated transporter trafficking is well documented, the cellular and molecular mechanisms governing transporter regulation and trafficking are not clearly defined. Given the pronounced effect pharmacological transporter blockade exerts on synaptic transmission, transporter sequestration is highly likely to significantly effect downstream neuronal signaling. Moreover, modulation of transporter availability is certain to have significant impact on the efficacy of psychoactive drugs. The major goals of this project are to elucidate the cellular and molecular mechanisms mediating acute transporter regulation and trafficking. Specifically, we hypothesize that (1) A brake mechanism controls constitutive and PKC-stimulated dopamine transporter (DAT) endocytosis; (2) Post-endocytic DAT trafficking differs from classical trafficking pathways; and (3) Amphetamine- and PKC-induced DAT sequestration are mechanistically distinct. These hypotheses are based on strong preliminary data demonstrating distinct endocytic motifs required for constitutive and PKC-regulated DAT internalization, novel DAT post-endocytic trafficking properties and PKC-independent properties of amphetamine-induced DAT sequestration. We will use site- directed mutagenesis to define constitutive, PKC-, and amphetamine-regulated DAT internalization and recycling signals. State-of-the-art live cellular imaging approaches will be used to define the mechanisms governing basal and regulated DAT endocytic trafficking. Finally, molecular and proteonomic approaches will define DAT-interacting proteins required for DAT endocytic trafficking. These approaches will provide a comprehensive picture of the mechanisms underlying regulated transporter trafficking. We expect our results will significantly impact future addiction and affective disorder treatment strategies. Moreover, the outcomes will greatly improve our understanding of the factors contributing to monoamine availability and signaling in the brain. PUBLIC HEALTH RELEVANCE: Mental illness and psychostimulant addiction are growing problems in the United States, reaching near epidemic proportions over the past several years. Despite numerous investigations into the mechanisms underlying these conditions, there are still large gaps in our knowledge. The current project will investigate the major targets in the brain for both psychostimulants and antidepressants, with the expectation that the forthcoming information may lead to novel therapeutic approaches for treating these disorders.